Abstract
The X-prolyl-dipeptidyl aminopeptidase from Streptococcus macedonicus
ACA-DC 191 was purified by anion exchange and hydrophobic interaction
chromatography. A single band of a molecular mass of about 84 000 g
mol
-1
appeared in SDS-PAGE; by gel filtration it was shown that the native
enzyme was dimeric. The enzyme showed optimum activity on
glycyl-prolyl-4-nitroanilide at pH 7.0, with a
= 0.42
mmol
L
-1 and a V
= 12.8
mol
mg
min
-1.
It was active over a temperature range of 10-60 °C. Over 60 °C, the enzyme
activity declined rapidly. The peptidase was completely inactivated
by PMSF, DTNB and Cu
2+, while metal chelators had no effect on enzyme
activity. By using the PCR technique with synthetic primers, the pepX
gene was amplified, cloned and sequenced. This 2 289 nucleotide gene
encodes a protein of 763 amino acids with a molecular mass of 86 866
g
mol
-1. The deduced amino acid sequence analysis of the pepX gene
shows a high identity with PepX enzymes from other lactic acid bacteria
and contains a motif around the active site serine (G-K-S-Y-L-G) that
is well conserved among the PepX enzymes.